EryP are the first differentiated cell type to form in the mammalian embryo and play a vital role in oxygen delivery and in generating shear forces necessary for normal vascular development. Despite their abundance and indispensable functions, the development and maturation of EryP remain poorly defined. Large, nucleated EryP arise within the blood islands of the yolk sac beginning ~E7.5 and begin to circulate around E9.5, when connections between the yolk sac and embryonic vasculature mature. Several days later, small cells of the definitive erythroid lineage (EryD) begin to differentiate within the fetal liver and enter the circulation, so that the two lineages are not easily distinguished. During the previous funding period, we developed transgenic mouse systems that allow the tagging and tracking of EryP and their nuclei throughout gestation. Major findings to emerge from this work were that EryP progress through previously unrecognized stages leading to their maturation, that they are a stable population present throughout gestation and do not gradually disappear, and that they accumulate transiently within the erythroblastic islands (EBIs) of the fetal liver (FL). Concomitant with EryP migration into the FL, a dramatic increase in adhesion molecule expression occurs along with significantly increased ability to bind fetal liver macrophages (FLMs). The ability of EryP to bind to FLMs is developmentally regulated, maximal during the window of time when they are found within the fetal liver, and partly dependent on VCAM-1. Large numbers of extruded EryP nuclei are found within the fetal liver at the time the first enucleated EryP are detected in the blood. EryP nuclei can be identified within FLMs after co- culture and in the native fetal liver, in vivo, suggesting that they are cleared and degraded by macrophages. After enucleation, the ability of circulating EryP to adhere to macrophages is lost and their numbers in the FL decline. We hypothesize that the fetal liver is a developmental niche for the maturation of primitive erythroblasts and that terminal steps in EryP maturation, including enucleation, occur in the EBIs of the fetal liver and involve adhesive interactions with macrophages. The fetal liver is just developing as EryP begin to circulate, around E9.5. Our observations therefore suggest a simple solution to the puzzling question of why enucleation of EryP is not detected until days after their appearance: terminal maturation, including nuclear extrusion, occurs in the fetal liver, which does not form until midgestation. The tools we have developed during the previous funding period will allow us to study the biology of primitive erythropoiesis at a resolution not previously possible. We propose to (1) determine whether macrophages provide a microenvironment for EryP maturation within the fetal liver;(2) evaluate the roles of integrins and their receptors in the maturation of primitive erythroblasts;and (3) investigate molecular events underlying the final stages of erythroid maturation. PUBLIC HEALTH RELEVANCE: Characterization of progenitor cell populations and elucidation of the common as well as the distinguishing features of embryonic versus adult erythroid development will be a prerequisite for the directed differentiation of human ES cells, HSCs or hematopoietic progenitors for therapeutic purposes in patients and for the efficient production of pure populations of red blood cells for transfusion. Pathways involved in erythroid development in the embryo may be dysregulated in leukemias and myelodysplastic disorders. The proposed studies should therefore be of broad biomedical significance.